qPCR analysis- can I use RQ? - (Dec/31/2011 )
I'm analyzing my first qPCR plate, and my intent has been to use relative quantification to find expression changes. The data I got from our PCR core specifies that the assay type was set to absolute quantification.
<*>Can an AQ assay be analyzed with RQ methods?
<*>What is the difference between AQ and RQ in terms of the actual qPCR run- i.e., what does the qPCR machine do differently?
You need standards of known copy number/concentration to do absolute quantification. You need calibrator and reference gene to do relative quantification. If you have both on a plate/s when you run it you can analyse both ways. Otherwise not. The machine measures fluorescence and Cts, analysation software modules are usually independent on the setting but you need to have WHAT to analyse.
I hope you just designed relative quantification experiment and included dilution series which were then mistaken as standards, because otherwise I honestly can't imagine how you could have RQ experiment, analysed as absolute. What exactly did you have on your assay?